Bio-ink development for three-dimensional bioprinting of hetero-cellular cartilage constructs.

Bioprinting is a promising tool to fabricate organized cartilage. This study aimed to investigate the printability of gelatin-methacryloyl/gellan gum (gelMA/gellan) hydrogels with and without methacrylated hyaluronic acid (HAMA), and to explore (zone-specific) chondrogenesis of chondrocytes, articular cartilage progenitor cells (ACPCs), and multipotent mesenchymal stromal cells (MSCs) embedded in these bio-inks.The incorporating of HAMA in gelMA/gellan bio-ink increased filament stability, as measured using a filament collapse assay, but did not influence (zone-specific) chondrogenesis of any of the cell types. Highest chondrogenic potential was observed for MSCs, followed by ACPCs, which displayed relatively high proteoglycan IV mRNA levels. Therefore, two-zone constructs were printed with gelMA/gellan/HAMA containing ACPCs in the superficial region and MSCs in the middle/deep region. Chondrogenic differentiation was confirmed, however, printing influence cellular differentiation.ACPC- and MSC-laden gelMA/gellan/HAMA hydrogels are of interest for the fabrication of cartilage constructs. Nevertheless, this study underscores the need for careful evaluation of the effects of printing on cellular differentiation.

Bone Morphogenetic Proteins for Nucleus Pulposus Regeneration.

Matrix production by nucleus pulposus (NP) cells, the cells residing in the center of the intervertebral disc, can be stimulated by growth factors. Bone morphogenetic proteins (BMPs) hold great promise. Although BMP2 and BMP7 have been used most frequently, other BMPs have also shown potential for NP regeneration. Heterodimers may be more potent than single homodimers, but it is not known whether combinations of homodimers would perform equally well. In this study, we compared BMP2, BMP4, BMP6, and BMP7, their combinations and heterodimers, for regeneration by human NP cells. The BMPs investigated induced variable matrix deposition by NP cells. BMP4 was the most potent, both in the final neotissue glysosaminoglycan content and incorporation efficiency. Heterodimers BMP2/6H and BMP2/7H were more potent than their respective homodimer combinations, but not the BMP4/7H heterodimer. The current results indicate that BMP4 might have a high potential for regeneration of the intervertebral disc. Moreover, the added value of BMP heterodimers over their respective homodimer BMP combinations depends on the BMP combination applied.

Allogeneic Mesenchymal Stem Cells Stimulate Cartilage Regeneration and Are Safe for Single-Stage Cartilage Repair in Humans upon Mixture with Recycled Autologous Chondrons.

Traditionally, mesenchymal stem cells (MSCs) isolated from adult bone marrow were described as being capable of differentiating to various lineages including cartilage. Despite increasing interest in these MSCs, concerns regarding their safety, in vivo behavior and clinical effectiveness have restrained their clinical application. We hypothesized that MSCs have trophic effects that stimulate recycled chondrons (chondrocytes with their native pericellular matrix) to regenerate cartilage. Searching for a proof of principle, this phase I (first-in-man) clinical trial applied allogeneic MSCs mixed with either 10% or 20% recycled autologous cartilage-derived cells (chondrons) for treatment of cartilage defects in the knee in symptomatic cartilage defect patients. This unique first in man series demonstrated no treatment-related adverse events up to one year postoperatively. At 12 months, all patients showed statistically significant improvement in clinical outcome compared to baseline. Magnetic resonance imaging and second-look arthroscopies showed completely filled defects with regenerative cartilage tissue. Histological analysis on biopsies of the grafts indicated hyaline-like regeneration with a high concentration of proteoglycans and type II collagen. Short tandem repeat analysis showed the regenerative tissue only contained patient-own DNA. These findings support the novel insight that the use of allogeneic MSCs is safe and opens opportunities for other applications. Stem cell-induced paracrine mechanisms may play an important role in the chondrogenesis and successful tissue regeneration found. Stem Cells 2017;35:256-264.

A Synthetic Thermosensitive Hydrogel for Cartilage Bioprinting and Its Biofunctionalization with Polysaccharides.

Hydrogels based on triblock copolymers of polyethylene glycol and partially methacrylated poly[N-(2-hydroxypropyl) methacrylamide mono/dilactate] make up an attractive class of biomaterials because of their biodegradability, cytocompatibility, and tunable thermoresponsive and mechanical properties. If these properties are fine-tuned, the hydrogels can be three-dimensionally bioprinted, to generate, for instance, constructs for cartilage repair. This study investigated whether hydrogels based on the polymer mentioned above with a 10% degree of methacrylation (M10P10) support cartilage formation by chondrocytes and whether the incorporation of methacrylated chondroitin sulfate (CSMA) or methacrylated hyaluronic acid (HAMA) can improve the mechanical properties, long-term stability, and printability. Chondrocyte-laden M10P10 hydrogels were cultured for 42 days to evaluate chondrogenesis. M10P10 hydrogels with or without polysaccharides were evaluated for their mechanical properties (before and after UV photo-cross-linking), degradation kinetics, and printability. Extensive cartilage matrix production occurred in M10P10 hydrogels, highlighting their potential for cartilage repair strategies. The incorporation of polysaccharides increased the storage modulus of polymer mixtures and decreased the degradation kinetics in cross-linked hydrogels. Addition of HAMA to M10P10 hydrogels improved printability and resulted in three-dimensional constructs with excellent cell viability. Hence, this novel combination of M10P10 with HAMA forms an interesting class of hydrogels for cartilage bioprinting.

Applicability of a newly developed bioassay for determining bioactivity of anti-inflammatory compounds in release studies--celecoxib and triamcinolone acetonide released from novel PLGA-based microspheres.

PURPOSE: To develop a bio-assay for measuring long-term bioactivity of released anti-inflammatory compounds and to test the bioactivity of celecoxib (CXB) and triamcinolone acetonide (TA) released from a new PLGA-based microsphere platform. METHODS: Human osteoarthritic chondrocytes were plated according to standardized procedures after batch-wise harvest and cultured for 3 days to prevent cell confluency and changes in cell behaviour. Prostaglandin E2 (PGE2) production stimulated by TNFα was used as a parameter of inflammation. A novel microsphere platform based on PTE-functionalised PLGA was used to incorporate CXB and TA. Loaded microspheres were added to transwells overlying the cells, with transfer of the wells to new cell cultures every 3 days. Inhibition of PGE2 production was determined over a period of 21 days. RESULTS: PLGA(75:25)-PTE microspheres were prepared and loaded with CXB and TA at 86 and 97% loading efficiency, respectively. In the bioactivity assay, PGE2 levels induced by TNFα were reduced to an average of 30% using microspheres loaded with 0.1 nmol CXB per transwell; with microspheres loaded with 0.1 nmol TA, PGE2 production was initially reduced to 3% and gradually recovered to 30% reduction. At 1 nmol loading, PGE2 was inhibited to 0-7% for CXB-loaded microspheres, and 0-28% for TA-loaded microspheres. CONCLUSIONS: We present a novel sustained release bioactivity assay which provides an essential link between in vitro buffer-based release kinetics and in vivo application. Novel PLGA-based microspheres loaded with TA and CXB showed efficient anti-inflammatory effects over time.

Participant selection for preventive Regenerative Medicine trials: ethical challenges of selecting individuals at risk.

The innovative field of Regenerative Medicine (RM) is expected to extend the possibilities of prevention or early treatment in healthcare. Increasingly, clinical trials will be developed for people at risk of disease to investigate these RM interventions. These individuals at risk are characterised by their susceptibility for developing clinically manifest disease in future due to the existence of degenerative abnormalities. So far, there has been little debate about the ethical appropriateness of including such individuals at risk in clinical trials. We discuss three main challenges of selecting this participant model for testing RM interventions: the challenge of achieving a proportional risk-benefit balance; complexities in the trial design in terms of follow-up and sample size; and the difficulty of obtaining informed consent due to the many uncertainties. We conclude that selecting the model is not ethically justifiable for first-in-man trials with RM interventions due to the high risks and uncertainties. However, the model can be ethically appropriate for testing the efficacy of RM interventions under the following conditions: interventions should be low risk; the degenerative abnormalities (and other risk factors) should be strongly related with disease within a short time frame; robust preclinical evidence of efficacy needs to be present; and the informed consent procedure should contain extra safeguards with regard to communication on uncertainties.

Osteoarthritis treatment using autologous conditioned serum after placebo.

BACKGROUND AND PURPOSE: Autologous conditioned serum (ACS) is a disease-modifying drug for treatment of knee osteoarthritis, and modest superiority over placebo was reported in an earlier randomized controlled trial (RCT). We hypothesized that when given the opportunity, placebo-treated patients from that RCT would now opt for ACS treatment, which would result in a greater clinical improvement than placebo. METHODS: Of 74 patients treated with placebo in the previous trial, 20 opted for ACS treatment. Patients who did not choose further treatment were interviewed about their reasons. Clinical improvement of the 20 ACS-treated patients was measured using knee-specific clinical scores, as was "response shift" at 3 and 12 months. RESULTS: In the 20 patients who did opt for ACS, the visual analog scale (VAS) score for pain improved; but after 12 months, clinical results were similar to those after placebo treatment. Response shift measurement demonstrated that the 20 patients had adapted to their disabilities during treatment. INTERPRETATION: Placebo-treated patients from an earlier trial were reluctant to undergo ACS treatment, in part due to the laborious nature of the therapy. In a subset of patients who opted for treatment, ACS treatment after placebo did not result in greater clinical improvement than placebo treatment only. However, due to the limited power of the current study and possible selection bias, definite advice on using or refraining from ACS cannot be given.

Missed low-grade infection in suspected aseptic loosening has no consequences for the survival of total hip arthroplasty.

BACKGROUND AND PURPOSE: Aseptic loosening and infection are 2 of the most common causes of revision of hip implants. Antibiotic prophylaxis reduces not only the rate of revision due to infection but also the rate of revision due to aseptic loosening. This suggests under-diagnosis of infections in patients with presumed aseptic loosening and indicates that current diagnostic tools are suboptimal. In a previous multicenter study on 176 patients undergoing revision of a total hip arthroplasty due to presumed aseptic loosening, optimized diagnostics revealed that 4-13% of the patients had a low-grade infection. These infections were not treated as such, and in the current follow-up study the effect on mid- to long-term implant survival was investigated. PATIENTS AND METHODS: Patients were sent a 2-part questionnaire. Part A requested information about possible re-revisions of their total hip arthroplasty. Part B consisted of 3 patient-related outcome measure questionnaires (EQ5D, Oxford hip score, and visual analog scale for pain). Additional information was retrieved from the medical records. The group of patients found to have a low-grade infection was compared to those with aseptic loosening. RESULTS: 173 of 176 patients from the original study were included. In the follow-up time between the revision surgery and the current study (mean 7.5 years), 31 patients had died. No statistically significant difference in the number of re-revisions was found between the infection group (2 out of 21) and the aseptic loosening group (13 out of 152); nor was there any significant difference in the time to re-revision. Quality of life, function, and pain were similar between the groups, but only 99 (57%) of the patients returned part B. INTERPRETATION: Under-diagnosis of low-grade infection in conjunction with presumed aseptic revision of total hip arthroplasty may not affect implant survival.

Inhibition of oncostatin M in osteoarthritic synovial fluid enhances GAG production in osteoarthritic cartilage repair.

Mediators in the synovial fluid are thought to play a major role in osteoarthritic cartilage turnover. The purpose of the current study was to investigate the role of oncostatin M (OSM) in osteoarthritis (OA) by evaluating the presence of the cytokine and its receptors in the OA joint and interfering with its activity in synovial fluid co-cultured with cartilage explants. OSM levels were increased in the synovial fluid of osteoarthritic patients compared to healthy donors. Immunohistochemistry confirmed the presence of both the leukaemia inhibitory factor (LIF) and OSM receptors for OSM throughout the whole depth of osteoarthritic cartilage and synovial tissue, whereas in healthy cartilage their presence seemed more restricted to the superficial zone. Blocking OSM activity, using an activity inhibiting antibody, in 25 % osteoarthritic synovial fluid added to OA cartilage explant cultures increased glycosaminoglycan (GAG) content from 18.6 mg/g to 24.3 mg/g (P < 0.03) and total production from 7.0 mg/g to 11.9 mg/g (P < 0.003). However, OSM exogenously added to cartilage explant cultures reflecting low and high concentrations in the synovial fluid (5 and 50 pg/mL) did not affect cartilage matrix turnover, suggesting that factors present in the synovial fluid act in concert with OSM to inhibit GAG production. The current study indicates the potential to enhance cartilage repair in osteoarthritis by modulating the joint environment by interfering with OSM activity.

Do monoblock cups improve survivorship, decrease wear, or reduce osteolysis in uncemented total hip arthroplasty?

BACKGROUND: Monoblock acetabular components used in uncemented total hip arthroplasty (THA) have certain mechanical characteristics that potentially reduce acetabular osteolysis and polyethylene wear. However, the degree to which they achieve this goal is not well documented. QUESTIONS/PURPOSES: The purpose of this study was to use a systematic review of controlled trials to test the hypothesis that monoblock cups have superior (1) polyethylene wear rate; (2) frequency of cup migration; (3) frequency of acetabular osteolysis; and (4) frequency of aseptic loosening compared with modular components used in uncemented THA. METHODS: A systematic search was conducted in the Medline, Embase, and Cochrane electronic databases to assemble all controlled trials comparing monoblock with modular uncemented acetabular components in primary THA. Included studies were considered "best evidence" if the quality score was either ≥ 50% on the Cochrane Back Review Group checklist or ≥ 75% the Newcastle-Ottawa quality assessment scale. A total of seven publications met our inclusion criteria. RESULTS: Best evidence analysis showed no difference in polyethylene wear rate, the frequency of cup migration, and aseptic loosening between monoblock and modular acetabular components. No convincing evidence was found for the claim that lower frequencies of acetabular osteolysis are observed with the use of monoblock cups compared with modular uncemented cups. CONCLUSIONS: The purported benefits of monoblock cups were not substantiated by this systematic review of controlled studies in that polyethylene wear rates and frequencies of cup failure and acetabular osteolysis were similar to those observed with modular implants. Other factors should therefore drive implant selection in cementless THA.

Non-viral gene therapy for bone tissue engineering.

The possibilities of using gene therapy for bone regeneration have been extensively investigated. Improvements in the design of new transfection agents, combining vectors and delivery/release systems to diminish cytotoxicity and increase transfection efficiencies have led to several successful in vitro, ex vivo and in vivo strategies. These include growth factor or short interfering ribonucleic acid (siRNA) delivery, or even enzyme replacement therapies, and have led to increased osteogenic differentiation and bone formation in vivo. These results provide optimism to consider use in humans with some of these gene-delivery strategies in the near future.

Coaxially electrospun scaffolds based on hydroxyl-functionalized poly(ε-caprolactone) and loaded with VEGF for tissue engineering applications.

The aim of this study was to fabricate nanofibrous scaffolds based on blends of a hydroxyl functionalized polyester (poly(hydroxymethylglycolide-co-ε-caprolactone), pHMGCL) and poly(ε-caprolactone) (PCL), loaded with bovine serum albumin (BSA) as a protein stabilizer and vascular endothelial growth factor (VEGF) as a potent angiogenic factor by means of a coaxial electrospinning technique. The scaffolds were characterized by scanning electron microscopy (SEM), fluorescence microscopy (FM), and differential scanning calorimetry (DSC). The scaffolds displayed a uniform fibrous structure with a fiber diameter around 700 nm. The release of BSA from the core of the fibers was studied by high performance liquid chromatography (HPLC), and it was shown that the coaxial scaffolds composed of blends of pHMGCL and PCL exhibited faster release than the comparative PCL scaffolds. VEGF was also incorporated in the core of the scaffolds, and the effect of the released protein on the attachment and proliferation of endothelial cells was investigated. It was shown that the incorporated protein preserved its biological activity and resulted in initial higher numbers of adhered cells. Thus, these bioactive scaffolds based on blends of pHMGCL/PCL loaded with VEGF can be considered as a promising candidate for tissue engineering applications.

Osteoarthritic synovial tissue inhibition of proteoglycan production in human osteoarthritic knee cartilage: establishment and characterization of a long-term cartilage-synovium coculture.

OBJECTIVE: Although both cartilage and synovium are affected in osteoarthritis (OA), no in vitro coculture models of human OA tissue have been described. The aim of this study was to develop an in vitro model that includes both the synovium and cartilage of patients with knee OA. METHODS: Explants of human OA cartilage and synovium were cultured alone or in coculture for 21 days. Histologic evaluation and analyses of lactate dehydrogenase release, matrix metalloproteinase (MMP) activity, content, release, and synthesis of glycosaminoglycan (GAG), and cytokine production were used to evaluate synovial tissue functionality and its effect on cartilage metabolism. To assess the possibility of intervention in the model system, the effect of triamcinolone was studied. RESULTS: Throughout the entire culture period, OA synovial tissue remained viable and produced cytokines. Monocultures of synovial and cartilage explants produced different cytokine subsets, with the subsets found in coculture being most similar to those previously described in OA synovial fluid. MMP activity was detectable only in the synovial explant monoculture and in coculture. Cocultures showed a reduction in final GAG content (P < 0.02), attributable to an inhibition of GAG production (P < 0.001) rather than an increase in GAG release. Addition of triamcinolone inhibited cytokine production and MMP activity in coculture and synovial tissue monoculture and counteracted the inhibition of GAG production induced by coculture. In cartilage monoculture, however, triamcinolone reduced GAG production. CONCLUSION: OA synovium affects cartilage metabolism by reducting GAG production. Triamcinolone can relieve this effect of synovial tissue, while being inhibitory when added to cartilage monoculture. These results clearly indicate the importance of tissue coculture as a promising tool for studying OA pathophysiology and for development of possible interventions.

Biomechanical assessment of the effects of decompressive surgery in non-chondrodystrophic and chondrodystrophic canine multisegmented lumbar spines.

PURPOSE: Dogs are often used as an animal model in spinal research, but consideration should be given to the breed used as chondrodystrophic (CD) dog breeds always develop IVD degeneration at an early age, whereas non-chondrodystrophic (NCD) dog breeds may develop IVD degeneration, but only later in life. The aim of this study was to provide a mechanical characterization of the NCD [non-degenerated intervertebral discs (IVDs), rich in notochordal cells] and CD (degenerated IVDs, rich in chondrocyte-like cells) canine spine before and after decompressive surgery (nucleotomy). METHODS: The biomechanical properties of multisegmented lumbar spine specimens (T13-L5 and L5-Cd1) from 2-year-old NCD dogs (healthy) and CD dogs (early degeneration) were investigated in flexion/extension (FE), lateral bending (LB), and axial rotation (AR), in the native state and after nucleotomy of L2-L3 or dorsal laminectomy and nucleotomy of L7-S1. The range of motion (ROM), neutral zone (NZ), and NZ stiffness (NZS) of L1-L2, L2-L3, L6-L7, and L7-S1 were calculated. RESULTS: In native spines in both dog groups, the greatest mobility in FE was found at L7-S1, and the greatest mobility in LB at L2-L3. Surgery significantly increased the ROM and NZ, and significantly decreased the NZS in FE, LB, and AR in both breed groups. However, surgery at L2-L3 resulted in a significantly larger increase in NZ and decrease in NZS in the CD spines compared with the NCD spines, whereas surgery at L7-S1 induced a significantly larger increase in ROM and decrease in NZS in the NCD spines compared with the CD spines. CONCLUSIONS: Spinal biomechanics significantly differ between NCD and CD dogs and researchers should consider this aspect when using the dog as a model for spinal research.

Hyaluronic acid and dextran-based semi-IPN hydrogels as biomaterials for bioprinting.

Bioprinting is a recent technology in tissue engineering used for the design of porous constructs through layer-by-layer deposition of cell-laden material. This technology would benefit from new biomaterials that can fulfill specific requirements for the fabrication of well-defined 3D constructs, such as the preservation of cell viability and adequate mechanical properties. We evaluated the suitability of a novel semi-interpenetrating network (semi-IPN), based on hyaluronic acid and hydroxyethyl-methacrylate-derivatized dextran (dex-HEMA), to form 3D hydrogel bioprinted constructs. The rheological properties of the solutions allowed proper handling during bioprinting, whereas photopolymerization led to stable constructs of which their mechanical properties matched the wide range of mechanical strengths of natural tissues. Importantly, excellent viability was observed for encapsulated chondrocytes. The results demonstrate the suitability of hyaluronic acid/dex-HEMA semi-IPNs to manufacture bioprinted constructs for tissue engineering.

Perivascular and diffuse lymphocytic inflammation are not specific for failed metal-on-metal hip implants.

BACKGROUND: Several studies suggest that histologic findings from tissues obtained at revision arthroplasty for failed metal-on-metal (MOM) total hip implants may reflect an immune reaction to particles or ions in some patients. However, only a limited number of cases without MOM implants were reported as controls in those studies. QUESTIONS/PURPOSES: The purpose of this study is to better define the extent and distribution of morphologic features attributed to an immune reaction in tissues sampled at revision surgery for failed nonMOM THA. PATIENTS AND METHODS: As part of a multicenter, prospective study, we reviewed 612 capsular and interface tissues obtained from 130 patients at revision THA. The samples were selected from periacetabular regions (154 samples from 103 patients), femoral implant/cement-bone interface (154 samples from 79 patients), and from areas of the joint capsule that had an intraoperative gross appearance suggesting the possibility of either infection or metallosis (256 samples from 129 patients). All patients had more than one sample obtained. The extent and distribution of lymphocytes and plasma cells, acute inflammation, and visible particles of debris were graded using criteria similar to those described to grade inflammation around failed MOM implants. RESULTS: We identified perivascular lymphocytes in 111 biopsy samples taken from 65 (50%) of 130 patients, and in 87 specimens from 57 (53%) of 107 patients thought to have aseptic loosening. Diffusely distributed lymphocytes were identified in 86 (66%) of 130 patients, and in 66 (62%) of the 107 hips with aseptic loosening, although few had the highest grade of inflammation. Increasing extent of diffuse and perivascular lymphocytes correlated with increasing extent of metal particles. CONCLUSIONS: Mild lymphocytic inflammation, diffuse and especially perivascular, is common in tissues around failed nonMOM implants. Although extensive inflammation in an inflammatory pseudotumor pattern is rare, it does occur in some cases of failed metal-polyethylene hip arthroplasties. The importance of inflammation is unknown, but the extent of diffuse inflammation shows a positive correlation with metal debris, so it could reflect a reaction to particles or ions in some patients.

Incidence of low-grade infection in aseptic loosening of total hip arthroplasty.

PURPOSE: We investigated the hypothesis that many total hip arthroplasty revisions that are classified as aseptic are in fact low-grade infections missed with routine diagnostics. METHODS: In 7 Dutch hospitals, 176 consecutive patients with the preoperative diagnosis of aseptic loosening of their total hip arthroplasty were enrolled. During surgery, between 14 and 20 tissue samples were obtained for culture, pathology, and broad-range 16S rRNA PCR with reverse line blot hybridization. Patients were classified as either not being infected, suspected of having infection, or infected according to strict, predefined criteria. Each patient had a follow-up visit after 1 year. RESULTS: 7 patients were classified as infected, 4 of whom were not identified by routine culture. 15 additional patients were suspected of having infection. 20 of these 22 patients received a cemented prosthesis, fixated with antibiotic-loaded bone cement. All 22 patients received prophylactic systemic antibiotics. 7 of them reported complaints one year after surgery, but only one showed signs of early loosening. However, additional surgery was not performed in any of the patients. INTERPRETATION: Although the proportions were not as high as previously reported in the literature, between 4% and 13% of patients with the preoperative diagnosis of aseptic loosening were infected. However, as thorough debridement was performed during surgery and prophylactic antibiotics were used, the diagnosis of infection did not have any obvious clinical consequences, as most patients performed well at the 1-year follow-up. Whether this observation has implications for long-term implant survival remains to be seen.

The influence of diffuse idiopathic skeletal hyperostosis on bone mineral density measurements of the spine.

OBJECTIVES: BMD has been described to be increased in patients with DISH. The contribution of the ossified anterior longitudinal ligament (ALL) on vertebral body BMD is currently unknown. We investigated the influence of DISH on BMD measurements using an experimental DXA scanning protocol. METHODS: Ten DISH specimens and 10 matched human cadaveric spines were used. After assessment of the localization and orientation of the ossified ALL with CT, BMD was measured using an experimental DXA protocol, exploiting the asymmetry of DISH in the thoracic spine. For controls, identical orientations were used and both groups were compared for differences in BMD. RESULTS: Specimens with DISH displayed a significantly higher BMD than their matched controls when the ossified ALL was present in the scanning field. Measurements of the left half of the spine were comparable for DISH specimens and controls (P = 0.446). The right-left difference in anteroposterior view was statistically significant within DISH specimens (P = 0.001), but not in controls (P = 0.825). CONCLUSIONS: The variability in measurements in different scanning orientations suggests a substantial contribution of the ossified ALL to the total BMD in DISH specimens, ranging from 23.6 to 39.0%. Vertebral body BMD does not seem to be increased, as demonstrated by comparable BMDs in the unaffected left half of the spine. It is suggested that routine anteroposterior DXA scanning may overestimate the true vertebral body BMD in DISH patients.

Evaluation of photocrosslinked Lutrol hydrogel for tissue printing applications.

Application of hydrogels in tissue engineering and innovative strategies such as organ printing, which is based on layered 3D deposition of cell-laden hydrogels, requires design of novel hydrogel matrices. Hydrogel demands for 3D printing include: 1) preservation of the printed shape after the deposition; 2) maintaining cell viability and cell function and 3) easy handling of the printed construct. In this study we analyze the applicability of a novel, photosensitive hydrogel (Lutrol) for printing of 3D structured bone grafts. We benefit from the fast temperature-responsive gelation ability of thermosensitive Lutrol-F127, ensuring organized 3D extrusion, and the additional stability provided by covalent photocrosslinking allows handling of the printed scaffolds. We studied the cytotoxicity of the hydrogel and osteogenic differentiation of embedded osteogenic progenitor cells. After photopolymerization of the modified Lutrol hydrogel, cells remain viable for up to three weeks and retain the ability to differentiate. Encapsulation of cells does not compromise the mechanical properties of the formed gels and multilayered porous Lutrol structures were successfully printed.